• Clinical and Experimental Reproductive Medicine
• /
• v.25 no.1
• /
• pp.59-64
• /
• 1998

This study was designed to evaluate the influence of pronuclear age on the survival and post-thawing development after cryopreservation of mouse embryos. Freezing and thawing were performed in the different pronuclear stages of mouse embryos after IVF. Embryos were obtained from $F_1$ hybrid mice and classified into 4 groups according to the pronuclear stage (6hr, 9hr, 12hr and 15hr after insemination). Pronuclear ova were slowly cooled in a biological freezer using 1.5M 1,2-propanediol and 0.1M sucrose as cryoprotectant. Thawing was done at room temperature and 1,2-propanediol was removed by multi-step dilutions. Both frozen-thawed embryos and control fresh embryos were cultured in vitro in Ham's F-10 medium supplemented with 4mg/ml BSA. In control group, the development rate after 48hr was 99.3%, and the complete hatching rate after 144hr was 61.3%. In experimental groups, the survival rate after thawing was 95.4% in 6hr, 88.7% in 9hr, 75.2% in 12hr and 62.4% in 15hr after insemination, the development rate after 48hr was 61.1, 77.0, 67.0 and 79.6%, respectively, and the complete hatching rate after 144hr was 25.7, 43.7, 42.2 and 60.0%, respectively. The survival rate in 15hr was significantly lower (p<0.05) compared with other groups. In vitro development rates after 48hr were similar in all groups, but complement hatching rate was significantly lower (p<0.05) in 6hr group. In conclusion, cryopreservation of mouse pronuclear ova with 2 distinct pronuclei (9hr and 12hr groups) showed better results after thawing compared with early (6hr group) or late pronuclear ova just prior to cleavage (15hr group).

• Plant Biotechnology Reports
• /
• v.2 no.2
• /
• pp.145-152
• /
• 2008

Aerides vandarum and Vanda stangeana are two rare and endangered vandaceous orchids with immense floricultural traits. The intergeneric hybrids were synthesized by performing reciprocal crosses between them. In vitro germination response of the immature hybrid embryos was found to be best on half-strength Murashige and Skoog medium supplemented with 20% (v/v) coconut water/liquid endosperm from tender coconut. Determination of hybridity was made as early as the immature seeds or embryos germinated in vitro, using randomly amplified polymorphic DNA (RAPD) markers. Out of 15 arbitrarily chosen decamer RAPD primers, two were found to be useful in amplification of polymorphic bands specific to the parental species and their presence in the reciprocal crosses. However, a decisive profile that can identify the reciprocal crosses could not be provided by RAPD. Amplification of the trnL-F non-coding regions of chloroplast DNA of the parent species and hybrids aided easy identification of the reciprocal crosses from the fact that maternal inheritance of chloroplast DNA held true for these intergeneric hybrids. Subsequent restriction digestion of the polymerase chain reaction (PCR) amplified trnL-F non-coding regions of chloroplast DNA also consolidated the finding. Such PCR-based molecular markers could be used for early determination of hybridity and easy identification of the reciprocal crosses.

• Korean Journal of Plant Tissue Culture
• /
• v.25 no.5
• /
• pp.319-322
• /
• 1998

Culture conditions for high frequency embryogenesis and plant regeneration in anther cultures of various F$_1$ hybrid and homozygous lines of pepper (Capsicum annuum L.) are described. Anthers pigmented less than halfway from the distal end were dissected from the flower bud in which petals elongated 2 mm higher than the receptacle. They were placed on Dumas medium supplemented with 0.1 mg/L 2,4-D and 0.1 mg/L kinetin. After four weeks of culture, embryos began to appear on anthers. After eight weeks of culture, frequencies of embryo formation reached up to 58.3%. Upon transfer to MS basal medium, greater than 95% of embryos developed into plantlets.

• Journal of Plant Biotechnology
• /
• v.33 no.4
• /
• pp.297-302
• /
• 2006

In general, the proliferation of orchids via somatic embryos has been used for mass production of somatic clones because of high propagation efficiency. In spite of high propagation rate, this method often brings somaclonal variation, especially polyploid frequency. Therefor we here concentrated to investigate the relationship between endopolyploidization patterns of explants and the occurrence of tetraploid variant in clonally proliferated Doritaenopsis via somatic embryo regeneration system. In the fully developed somatic embryo, upper part contained 2C to 16C while middle and lower parts showed 2C to 32C DNA content. Two-week-old embryo contained 2C to 16C, whereas those regenerated after 4 to 10-week-old contained 2C to 64C nuclei. Results showed that endoreduplication was variable depending upon tissue types, ages, and parts in one species. lower part of somatic embryo having high endoreduplication degree increased the regeneration of tetraploid variants by about 3-fold comparing to upper part of somatic embryo culture. polyploid frequency occurrence might be closely related to the high levels of endoreduplication of somatic embryos used as explant. It suggested that the upper part of somatic embryo having comparatively low endoreduplication degree is suitable for the stable in vitro propagation system.

• FLOWER RESEARCH JOURNAL
• /
• v.17 no.4
• /
• pp.221-225
• /
• 2009

Interspecific crossing was conducted to obtain hybrid lilly with better quality of adapting to the unfavorable environment using oriental Lilium spp. as a female parent. Average interspecific crossing rate between oriental and asiatic lines by cut-style pollination was as low as 29%. Although the average germination rate of zygotic hybrid embryo between oriental 'Rodolfa' ${\times}$ asiatic 'Toronto' was increased from 76.6% to 78.3%. on 114 or 1/6 strength MS medium compared to the control, the rate of zygotic embryos formation between oriental 'Farolito' ${\times}$ asiatic 'Buff Pixie' was significantly enhanced up about 86~90% on 1/2 strength MS medium. Meanwhile, germination rate of interspecific hybrid embryo between oriental 'Snow Cristal' ${\times}$ asiatic 'Royal Trinity' showed up 100%. Germination rate of interspecific hybrids slightly increased by addition of $0.5-1.0mg{\cdot}L^{-1}$ GA. Germination rate of zygotic embryos between oriental 'Farolito' ${\times}$ asiatic 'Buff Pixie' and oriental 'Belcanto' ${\times}$ L. callosum was highest on the MS medium containing $0.01mg{\cdot}L^{-1}$ NAA and $0.1mg{\cdot}L^{-1}$ kinetin, as 66.6% and 45.2% respectively.

• Korean Journal of Plant Tissue Culture
• /
• v.24 no.4
• /
• pp.239-256
• /
• 1997

Plants belonging to the category of 2n apomixis or agamospermy form embryos and seeds without the processes of normal meiosis and syngamy. Seeds produced in this way have identical genotype of their maternal parent. Three different types of agamospermy are recognized: diplospory, apospory, and adventitious (adventive) embryony. $F_1$ hybrid cultivars cannot be used as seed sources in the next ($F_2$) generation because this generation would be extremely variable as a result of genetic segregation. Hybrid vigor is also reduced in the $F_2$ generation. Therefore, parental stocks for hybrid seed production need to be maintained and cross must be continuously repeated. Agamospermic 2n apomixis would make it possible to fix the genotype of a superior variety so that clonal seeds faithfully representing that genotype could be continuously and cheaply produced independent of pollination. That is, $F_1$ hybrid seeds could be produced for many generations without loss of vigor or genotype alteration. Production of apomictic $F_1$ hybrid seed would be simplified because line isolation would not be necessary to produce seed or to maintain parental lines, and the use of male-sterile lines could be avoided. Overall, apomixis would enable a significant reduction in hybrid seed production costs. Additionally, the production of clonal seed is not only important for seed propagated crops, but also for the propagation of heterozygous fruit trees and timbers. Clonal seed would help avoid costly and time-consuming vegetative propagating methods that are currently used to ensure the large-scale production of these plants. Apomixis is scattered throughout the plant kingdom, but few important agricultural crops possess this trait Therefore, most research to date has centered on introgressing the trait of apomixis into agricultural crops such as wheat, maize, and some forage grasses from wild distant relatives by traditional cross breeding. The classical breeding approach, however is slow and often impeded by many breeding barriers. These problems could be surmounted by taking mutagenesis or molecular approach. Arabidopsis thaliana is a tiny sexually reproducing plant and is convenient in constructing and screening in molecular researches. Male-sterile mutants of Arabidopsis are particularly suitable genetic background for mutagenesis and screening for apomictic mutants. Molecular approaches towards isolating the genes controlling the apomictic process are feasible. Direct isolation of genes conferring apomixis development would greatly facilitate the transfer of this trait to wide variety of crops. Such studies are now in progress.

• Clinical and Experimental Reproductive Medicine
• /
• v.29 no.1
• /
• pp.1-12
• /
• 2002

Objective: In order to acquire the technique for the establishment of human embryonic stem cells (ESe) derived from the human frozen-thawed embryos produced in IVF-ET program, this study was performed to establish mouse ESC derived from the in vitro fertilized embryos. Materials and Methods: After Fl hybrid (C57BL female $\times$ CBA mael) female mice were superovulated with PMSG and hCG treatment, their oocytes were retrieved and inseminated, and the fertilized embryos were cultured for 96-120 hours until the expected stages of blastocysts were obtained. To isolate the inner cell mass (ICM), either the blastocysts were treated with immunosurgery, or the whole embryos were cultured for 4 days. Isolated ICMs were then cultured onto STO feeder cell layer, and the resultant ICM colonies were subcultured with trypsin-EDTA treatment. During the subculture process, ESC-like cell colonies were observed with phase contrast microscopy. To identify ESC in the subcultured ESC-like cell colonies, alkaline phosphatase activity and Oct-4 (octamer-binding transcription factor-4) expression were examined by immunohistochemistry and RT-PCR, respectively. To examine the spontaneous differentiation, ESC-like cell colonies were cultured without STO feeder cell layer and leukemia inhibitory factor (LIF). Results: Seven ESC-like cell lines were established from ICMs isolated from the in vitro fertilized embryos. According to the developmental stage, the growth of ICMs isolated from the expanded blastocysts was significantly better than that of ICMs isolated from the hatched blastocysts (80.3% vs. 58.7%, p<0.05). ESC-like cell colonies were only obtained from ICMs of expanded blastocysts. However, the ICMs isolated from the embryos treated with immunosurgery were poorly grown and frequently differentiated during the culture process. The established ESC-like cell colonies were positively stained with alkaline phosphatase and expressed Oct-4, and their morphology resembled that observed in the previously reported mouse ESC. In addition, following the extended in vitro culture process, they maintained their expression of cell surface markers characteristic of the pluripotent stem cells such as alkaline phosphatase and Oct-4. When cultured without STO feeder cell layer and LIF, they were spontaneously differentiated into the various types of cells. Conclusion: The findings of this study suggest that the establishment of mouse ESC can be successfully derived from the in vitro fertilized embryos. The established ESC-like cells expressed the cell surface markers characteristic of the pluripotent stem cells and spontaneously differentiated into the various types of cells.

• Journal of Plant Biotechnology
• /
• v.30 no.4
• /
• pp.353-357
• /
• 2003

We studies seed set in the interspecific F1 /backcross hybrids of Lilium species. In the interspecfic hybrid of L. longiflorum cv. Gelria with L.${\times}$ fomolongi cv. Raizan,93％ fruit set was obtained by stigmatic pollination in comparison to 53％ from cut-style pollination. Accordingly, the number of seed set resulting from stigmatic and cut-style pollination was 147 and 53, respectively. Pollination o( both stigmatic and cut-style pollination resulted in 47％ fruit set in the hybrid of L. longiflorum cv. Lorina with L.${\times}$ fomolongi cv. Raizan. However, stigmatic pollination formed 413 seeds, whereas only 24 seeds were obtained by cut-style pollination in this cross. The hybrid of L.${\times}$ fomolongi cv. Raizan with L. longiflorum cv. Come set 40％ fruit with a total of 43 seeds by stigmatic pollination. However, no fruit set was observed in cut-style pollination in this hybrid. Backcrossing the F1 hybrid by cut-style pollination of L. longiflorum cv. Lorina ${\times}$ Asiatic hybrid cv. Chicago with the latter parent led to 53％ fruit set, and 109 embryos were obtained. Likewise, backcrossing following cut-style pollination of L. longiflorum cv. Lorina ${\times}$ Asiatic hybrid cv. Corsia with the latter parent formed 67％ fruits and 107 embryos. However, in the remaining interspecific hybrids, cut-style pollination set no fruit.

• The Korean Journal of Zoology
• /
• v.32 no.3
• /
• pp.258-263
• /
• 1989

Nuclear Transplantation between Rana pipiens and Rana dybowskii When diploid blastula nuclei of Rana pipiens are traraplanted into enucleated eggs of Rana dybowskii the resulting nucleocytoplasmic hybrids are lethal-those development were arrested around the stage of the dorsal lip formation For the improvement of developmental capacity, serial nuclear transplantation was carried out. Even though serial transplantation of 15 generations showed normal development in each generation until gastrula stage, there was no sign of fundamental improvement in development afterward. This results implied that up to gastrulation normal DNA replication and cell division can take place in foreign cytoplasm. Since chromosomal aberrations both in shape and number were usually observed, the nuclei must have been modifted while resided in the foreign cytoplasm. Those nuclei didn't participate in normal development and led the embryos to early death. Tissue graft experiment indicated that the abnormal behavior of this lethal nucleocytoplasmic hybrid is an inherent property which is not corrected by the contact with its own tissue.

• Korean Journal of Animal Reproduction
• /
• v.19 no.3
• /
• pp.227-234
• /
• 1995

This study was carried out to investigate the effects according to the time course of glucose exposure on the development of one-cell embryos beyond morula in CR$_laa$ medium. One-cell zygotes from B6CBA F$_1$ mice were recovered at 24 ~ 25h after hCG and cumulus cells were removed with 0.1% hyaluronidase. The embryos were pooled and subsequently divided into each groups and cultured in CR$_laa$ at 37$^{\circ}C$ in 5% CO$_2$ in aIr. The embryos were either, placed in CR$_laa$ containing various concentration (5.5, 16.5, 27.5 and 38.5 mM) of glucose for 1 min. and subsequently returned to the fresh culture medium (without glucose), or were transferred to the same media containing glucose at 72 h post hCG. The results obtained in these experiments were summarized as follows: 1. The development rates of zygotes, recovered from the oviducts in M2 and cultured in CR$_laa$ with 3mg/Im FAF-BSA, to expanded blastocysts (25.7%) and hatching bIastocysts (17.6%) were significantly higher than those of zygotes recovered in TL Hepes (0% and 0%, respectively). 2. The development rates of one-cell embryos exposed to 27.5 mM glucose at 72 h post hCG for 1 min, were 68.8% (CR$_1$+BSA) a and 77.1% (CR$_1$+FBS) of expanded blastocyst stage, but there were no significant differences between the embryos exposed for 1 min. or transferred at 72 h. 3. Regardless of glucose concentration (5.5, 16.5, 27.5 & 38.5mM), 45.7~61.5% of embryos developed to the blastocyst stage. There were no significant differences between any of the treatments on the devel-opment of one-cell embryos. Therefore, the detrimental effect of highly concentration was not appeared.